The application of Adenoviruses (Ad) as vectors for therapeutic gene delivery, vaccines, or cancer therapy is a very promising approach for treatment of a variety of highly prevalent and clinically important diseases. Ad vectors form highly stable proteinaceous particles that can efficiently deliver therapeutic transgenes into both dividing and quiescent cells. Other advantages of Ad vectors as gene therapy tools include: capacity for delivery of large therapeutic genes, the ease of genetic manipulation, the ability to produce large quantities of clinical grade vectors, and the episomal nature of the vector genome. However, the successful application of Ad vectors for gene therapy applications in clinical settings has been hampered by the unexpected complexity of the vector-host interactions. Interactions between the Ad particles and the cell and factors of the immune system in humans may result in severe virus-induced toxicity and therefore dire clinical outcomes. One of the harshest setbacks in Ad vector therapeutic development was caused by the death of a patient enrolled in a clinical trial in 1999 (Raper et al., 2003). After infusion of Ad vector into hepatic artery, the patient died due to the severe Ad vector-induced inflammatory response, which resulted in multiple organ failure, disseminated intravascular coagulation, and a cytokine storm (Raper et al., 2003; Raper et al., 2002). Clearly, detailed and thorough understanding of the host response to intravascular Ad vector administration is a crucial prerequisite for the successful and safe use of Ad vectors in humans.
Our previous studies indicate that the activation of pro-inflammatory cytokines and chemokines by tissue residential macrophages in vivo occurs downstream of the IL-1α-IL-1RI signaling pathway and is triggered by adenovirus Arginine-Glycine-Aspartic acid, RGD, motif-dependent binding to macrophage β3 integrins (Di Paolo et al., 2009). The RGD motif, located within a flexible and highly variable loop of the adenovirus penton base protein, which is considered the functional moiety of the capsid that facilitates virus internalization into cells by promoting integrin clustering at the sites of virus attachment and viral endocytosis. Accordingly, RGD motif-deleted viruses demonstrate reduced efficacy of internalization into cells, a feature that is not conducive to efficient vector-mediated gene transfer.
In vitro studies demonstrated that Ad infection starts with the virus binding to a high affinity primary attachment receptor on the cell surface (Nemerow, 2000). The trimeric Ad fiber protein mediates this interaction when its distal knob domain binds to a specific cellular receptor. For binding to cells, species A, C, D, E, and F human Ad may utilize the coxsackievirus and Ad receptor (CAR) (Roelvink et al., 1998); however, the majority of human species B Ads utilize CD46 as a high affinity cellular attachment receptor (Gaggar et al., 2003). In this regard, soluble Ad fiber or anti-fiber antibodies can inhibit infection by the Ad.
Fiber-mediated binding of Ad to cells is followed by RGD motif-mediated binding of the viral penton base protein to cellular integrins (e.g., αvβ3 and αvβ5) (Nemerow and Stewart, 1999). This interaction induces integrin activation and cytoskeleton rearrangement that facilitates internalization of the virus particle into the cell. Therefore, Ad penton interaction with cellular integrins is necessary for efficient virus entry into the cell, cell transduction and gene transfer to occur. Based in part on this knowledge, previous disclosure provided Ad vectors that were designed to change Ad interactions with integrins in the in vitro cell culture systems. See U.S. Pat. No. 5,712,136, which is incorporated by reference herein. However, in the in vitro systems, such as 293 cell line, which is widely used for growing of newly constructed Ad vectors and for preparation of high-titer vector stocks suitable for pre-clinical and clinical applications, Ad interactions with integrins play insignificant role in supporting virus cell infection. Indeed, Ad mutants, which are deleted for and lack RGD amino acid motif in penton protein (AdΔRGD, refs) can be grown to high titers and are efficiently propagated on 293 cells in vitro (Shayakhmetov et al., 2005). Importantly, the U.S. Pat. No. 5,712,136 does not teach of constructing Ads for in vivo applications, and capable of triggering reduced inflammatory responses and transducing cells in the body after intravascular administration.
For successful application of Ad vectors for correcting human genetic diseases and for the treatment of disseminated and localized metastatic cancers, Ad vectors with improved safety profiles are critically needed. Furthermore, what is needed are new Ad vectors that exhibit efficient cell transduction and reduced activation of inflammation after intravascular injection.